Monocyte recruitment and macrophage regulation of tumor growth and angiogenesis

描述(由申请人提供)：近期和长期的职业目标：我近期的职业目标是进一步发展我在肿瘤免疫学方面的技能，主要是利用各种技术设计和进行我自己的实验。此外，我的目标也是学习利用我现有的资源，包括设备和人员，以进一步了解单核细胞和巨噬细胞在肿瘤进展中的作用。我的长期职业目标是在一家学术机构获得一个教员的职位，这样我不仅有机会继续进行基础研究，还可以将我的发现应用到家畜的癌症临床试验中。此外，我希望有能力教授学生肿瘤免疫学，因为它与兽医有关。关键要素研究职业发展计划：我的职业发展计划有多个关键要素。首先，也是最重要的，是我在陶氏和汉森博士的指导下，他们加起来在指导博士后方面有相当多的经验。其次，CSU的动物癌症中心提供了大量的指导支持，并为协作和科学工作提供了开放的政策。这个设施有现代化的设备，包括多个流式细胞仪，还有足够的空间让我进行研究。动物癌症中心的项目通过许多研讨会和杂志俱乐部得到加强，我计划参加这些活动，教职员工和学生定期参加。这些是分享想法和形成合作的平台。最后，Dow博士和我与科罗拉多大学健康科学中心和国家犹太医学和研究中心的密切关系使我们能够进一步合作，并接触到人类医学和其他免疫学研究领域。这些研究所是免疫学研究的领先机构，因此我计划最大限度地利用这一资源。项目简介：肿瘤相关巨噬细胞()在促进肿瘤生长中起着至关重要的作用。被认为主要来源于单核细胞，这些单核细胞通过各种信号(如CCL2和CSF1)主动募集到肿瘤中。然而，文献中的数据表明，也可以由中性粒细胞(PMN)引起，这使得他们也是一个有吸引力的人群来研究。一旦进入肿瘤，单核细胞，可能还有中性粒细胞，分化成巨噬细胞，具有强大的免疫抑制以及肿瘤生长和促进血管生成的特性。然而，目前对哪些骨髓来源的细胞被招募到肿瘤中以及肿瘤炎症在这一过程中所起的作用缺乏很好的了解。这种募集的动力学也不清楚，单核细胞/中性粒细胞分化为免疫抑制因子的过程是否可逆。因此，假设肿瘤炎症是CCL2/CSF1产生和单核细胞及中性粒细胞募集的主要驱动因素，通过抑制炎症、耗尽单核细胞和/或中性粒细胞或激活新来的巨噬细胞来阻断这种募集可以显著抑制这些的积聚，从而抑制肿瘤生长。这些假设将有三个具体的目标。目的1评估肿瘤炎症对肿瘤CCL2和CSF-1产生的影响，以及这些因素在单核细胞/中性粒细胞募集和分化为TAM中的作用。这些实验将利用缺乏CCL2或其受体的小鼠和缺乏生物活性的CSF-1的小鼠来检验这些因素对单核细胞募集的作用。此外，当PMN募集被阻断时，将利用小鼠IL-8(KC)和IL-8受体基因敲除小鼠的抗体耗竭来检测肿瘤的变化。在这些小鼠模型中，将进行微珠标记实验，以跟踪肿瘤中单核细胞和中性粒细胞的流入和周转速度。目的研究单核细胞和中性粒细胞耗竭-氯屈膦酸盐脂质体或抗体介导的中性粒细胞耗竭对在肿瘤中蓄积和肿瘤生长的影响。目的3使用一种有效的天然免疫激活剂，评价免疫刺激对单核细胞和中性粒细胞的影响，以及对肿瘤内抑制的群体的激活和发展。此外，这种先天免疫刺激和脂质体氯膦酸盐耗竭的结合将被作为逆转免疫抑制表型的新机制进行研究。这些研究将为了解的生物学以及肿瘤如何调控他们的招募和分化提供重要的见解。这些信息反过来将有助于设计治疗策略，以克服肿瘤固有的免疫抑制环境。

DESCRIPTION (provided by applicant): Immediate and Long term career goals: My immediate career goals are to further develop my skills in tumor immunology, mainly in designing and carrying out my own experiments using a wide variety of techniques. In addition, my goals are also to learn to utilize my resources available, both equipment and personnel in order to further our understanding of the role of monocytes and macrophages in tumor progression. My long term career goal is to obtain a faculty position at an academic institution which offers me the opportunity to not only continue conducting basic research but also allow for application of my findings to cancer clinical trials in domestic animals. In addition, I wish to have the ability to teach students about tumor immunology as it relates to veterinary medicine. Key elements research career development plan: There are multiple key elements to my career development plan. First, and foremost, is my mentoring under Drs. Dow and Henson, who combined have considerable experience in mentoring post-doctoral fellows. Secondly, the Animal Cancer Center at CSU provides plenty of mentoring support as well as an open door policy for collaborative, scientific work. The facility has modern equipment, including multiple flow cytometers, and plenty of space for me to conduct my research. The program at the Animal Cancer Center is enhanced by many seminars and journal clubs, which I plan to attend and are well-attended by faculty and students on a regular basis. These are platforms for which ideas are shared and collaborations formed. Lastly, Dr. Dow's and my strong relationship with the University of Colorado Health Sciences Center and National Jewish Medical and Research Center allows for even further collaboration as well as exposure to human medicine and other areas of immunological research. These institutes are among the leading institutions for immunology research, thus I plan to use this resource to its fullest potential. Project Summary: Tumor associated macrophages (TAM) play an essential role in promoting tumor growth. TAM are thought to arise primarily from monocytes that are actively recruited to the tumor by various signals (i.e. CCL2 and CSF-1). However, data available in the literature suggests that TAM can also arise from neutrophils (PMN), making them an attractive population to examine as well. Once in the tumor, monocytes, and possibly PMN, differentiate into macrophages with potent immunosuppressive as well as tumor growth and angiogenesis-promoting properties. However, at present there lacks both a good understanding of which populations of bone marrow derived cells are recruited to the tumors and the role that tumor inflammation plays in the process. Nor are the kinetics of this recruitment understood or whether the differentiation of monocytes/PMNs into immunosuppressive TAM is reversible. Therefore, it is hypothesized that tumor inflammation is a major driver of CCL2/CSF-1 production and monocyte and PMN recruitment, and that interrupting this recruitment by suppressing inflammation, depleting monocytes and/or PMN, or activating newly arrived macrophages can significantly suppress the accumulation of these TAM and inhibit tumor growth. These hypotheses will be addressed with 3 specific aims. Aim 1 will assess the effects of tumor inflammation on tumor CCL2 and CSF-1 production and the role of these factors in monocyte/PMN recruitment and differentiation into TAMs. These experiments will utilize mice lacking CCL2 or its receptor and mice deficient in biologically active CSF-1 to examine the role of these factors on monocyte recruitment. In addition, antibody depletion of mouse IL-8 (KC) and IL-8 receptor knockout mice will be utilized to examine tumor changes when PMN recruitment is blocked. Within these mouse models, microbead labeling experiments will be undertaken to track the influx and turnover rate of monocytes and PMN in tumors. Aim 2 addresses the effects of monocyte and PMN depletion-using liposomal clodronate or antibody-mediated PMN depletion on accumulation of TAM in the tumor and tumor growth. Aim 3 involves use of a potent activator of innate immunity to assess the effects of immune stimulation on monocytes and PMN as well as TAM activation and development of suppressive TAM population within tumors. In addition, the combination of this innate immune stimulus and liposomal clodronate depletion will be investigated as a novel mechanism of reversing the TAM immunosuppressive phenotype. These studies will provide important insights into the biology of TAM and how their recruitment and differentiation is regulated by tumors. This information will in turn be useful in designing therapeutic strategies to overcome the inherently immunosuppressive environment of tumors.